Currently, there is no reliable commercially available cell-free nucleic acid quantification approach for estimating woman ovarian reserve, predicting woman stimulation response, IVF outcome and no therapy targeting these cell-free nucleic acids to improve ART success.
In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) data, clearly indicates disappointing results, even in the most successful programs. Indeed, in France, the ART success rate ranges from 25% to 28% in terms of live birth per oocyte retrieval. Some defects leading to problems with embryo implantation are still unexplained and the presence of abnormal levels of cell-free nucleic acids in blood could constitute another area of unexplained infertility.
During follicular development, oocytes are in close contact with the surrounding cumulus cells (CCs) to form the cumulus-oocyte complex (COC). The crosstalk between oocytes and CCs occurs through gap junctions (Albertini et al., 2001). This paracrine signaling is crucial for the acquisition of developmental competence in oocytes and CCs (Gilchrist et al., 2008). These reciprocal regulations are carefully modulated by some key genes that are themselves regulated by miRNAs (Assou et al., 2013). MicroRNAs belong to the “small RNA” family and are evolutionarily conserved from invertebrates to vertebrates (Lagos-Quintana et al., 2001). MiRNAs were first identified in Caenorhabditis elegans at the beginning of the nineties (Lee et al., 1993). They are non-coding single-stranded RNA molecules of 19-25 nucleotides in length that arise from intergenic or intragenic genomic regions. In mammals, miRNAs are usually complementary to a small region in the 3′ UTR (untranslated region) of messenger RNAs (mRNAs).
Some miRNAs are found in body fluids. As they are contained in exosomes, they are highly stable in body fluids because protected from RNAses. The potential use of these circulating miRNAs as novel, non-invasive diagnostic/prognostic biomarkers is the focus of many investigations (Mitchell et al., 2008) and they are already used as biomarkers for the diagnosis and prognosis of several gynecological and pregnancy disorders (Carletti et al., 2009). MicroRNAs (miRNAs) are small (19-25 nucleotides), single-stranded, non-coding RNA molecules that bind specifically to and post-transcriptionnally regulate several messenger RNAs (mRNAs) (Thomas et al., 2010). MiRNAs play important physiological roles and miRNA dysregulations can lead to pathologies. In fertility, miRNAs are associated with the functional regulation of gonadal somatic cells (Leydig and Sertoli cells in testis, and granulosa and cumulus cells in the ovary) involved in steroid synthesis. For example, in male mice, deletion of Dicer (a protein essential for miRNA maturation) in Sertoli cells leads to infertility due to the complete absence of spermatozoa and progressive testicular degeneration (Hossain et al., 2012). In female mice, Dicer invalidation leads to infertility due to multiple defects in ovarian functions, including abnormal cycles and abnormal response to gonadotropin leading to ovulation problem (Nagaraja et al., 2008). MiRNAs could have a major role in the regulation of follicular cell functions, such as steroidogenesis, apoptosis, luteinization, as well as in ovulation process (Hawkins et al., 2010). For instance, treatment of mouse mural granulosa cells with luteinizing hormone leads to the deregulation of a set of miRNAs (particularly miR-132 and miR-212 overexpression) that are possibly important for the control of ovarian functions (Fiedler et al., 2008). Overexpression of miR-93 could disturb ovary development. Indeed, miR-93 targets the mRNA encoding LHX8, a protein that contains a Lim homeodomain required for the transition from primordial to primary follicle (Pangas et al., 2006).
Many studies have shown that hormones from the hypothalamic-pituitary-gonadal axis, which are essential for sexual maturation and reproductive function in mammals, are also involved in the regulation of some miRNAs. Gonadotropin-Releasing Hormone (GnRH) stimulates the synthesis and the secretion of the pituitary gonadotropins Luteinizing hormone (LH) and Follicle Stimulating Hormone (FSH) that then regulate the production of gonadal steroids and gametogenesis (Conn et al., 1994; Kaiser et al., 1997). GnRH also induces the expression of multiple miRNAs, particularly miR-132 and miR-212, which are encoded by the same gene that is induced by GnRH (Godoy et al., 2011). LH acts on ovarian granulosa cells to induce ovulation and luteinization, resumption of oocyte meiosis and cumulus cell expansion that are crucial steps for adequate ovulation. Moreover, LH acts as a survival factor by preventing apoptosis of granulosa cells (Robker et al., 1998; Chaffin et al., 2001). Interestingly, LH also up-regulates miR-132, miR-212 and miR-21 in mural granulosa cells (Fiedler et al., 2008). MiR-21 is overexpressed in many tumors, including breast, pancreatic, colorectal and oesophageal cancer, and thus is considered as an oncomiRNA (Cho et al., 2007; Verghese et al., 2008; Dillhoff et al., 2008). MiR-21 depletion induces caspase-dependent apoptosis of mouse granulosa cells in vitro and in vivo (Carletti et al., 2010), highlighting the physiological anti-apoptotic role of miR-21 in normal tissues. MiR-200b and miR-429 depletion inhibits LH synthesis by repressing transcription of the gene encoding the subunit of LH. This results in lower serum LH concentration and absence of the LH surge, leading to ovulation failure (Hasuwa et al., 2013). Thus, the hypothalamus-pituitary-ovarian axis requires miR-200b and miR-429 to ensure ovulation. Finally, miR-122 is involved in the down-regulation of LH receptor expression by increasing the expression of LH receptor mRNA Binding Protein (LRBP) via activation of SREBPs (Azhar et al., 2013; Menon et al., 2013).
FSH has a crucial role both in follicle development and granulosa cell proliferation and differentiation. Several miRNAs, including miR-143, miR-125b, miR-21 and the let-7 family, are involved in follicular development in the mouse (Yao et al., 2009). The expression of these RNAs is very low in primordial follicles, but they become readily detectable in granulosa cells of primary, secondary and antral follicles. MiR-143, let-7a and miR-15b are negatively regulated by FSH (Yao et al., 2009). Moreover, miR-133b is involved in FSH-induced estrogen production, by binding to the 3′UTR of Foxl2 and thus reducing FOXL2 protein level in granulosa cells (Dai et al., 2013). FOXL2 is expressed in the ovaries and is necessary for granulosa cell function (Schmidt et al., 2004), particularly through regulation of steroidogenesis genes, including StAR and CYP19A1 that are essential for promoting estradiol production (Pisarska et al., 2011; Caburet et al., 2012).
The implication of miRNAs in the hormonal regulation during folliculogenesis and in the oocyte-niche crosstalk could be exploited for identifying new non-invasive biomarkers of fertility. Moreover, the development of therapies that block the expression or mimic the functions of specific miRNAs could represent a new therapeutic strategy for many gynecological disorders. Similarly, cell-free DNA (cfDNA) molecules, which are released mostly by apoptotic or necrotic cells, are also found in fluids and can be used as biomarkers of pathological conditions (Schwarzenbach et al., 2011). Indeed, cfDNA has been detected in human semen (Chou et al., 2004). This cell-free seminal DNA contains DNA epigenetic information that is essential for proper spermatogenesis (Wu et al., 2013). Circulating cfDNA in the bloodstream is also used to detect gynecological abnormalities and fetal cfDNA in maternal blood constitutes a non-invasive biomarker for fetal aneuploidy (Lo et al., 1999; Bischoff et al., 2002; Bischoff et al., 2005; Bauer et al., 2006; Lo et al., 2008; Wright et al., 2009; Lambert-Messerlian et al., 2013). Recently, it was reported that increased plasma cfDNA levels are associated with low pregnancy rates in IVF programs (Czamanski-Cohen et al., 2013). However, the only correlation was between cfDNA and pregnancy outcome, once the patient was pregnant.
There is no known method for assessing the ovarian reserve of individual patient, therefore determination of ovarian reserve is important in the treatment of infertility.